An in vitro evaluation of human cytochrome P450 3A4 inhibition by selected commercial herbal extracts and tinctures

Phytomedicine, Vol. 7(4), pp. 273-282 © Urban & Fischer Verlag 2000 http://www.urbanfischer.de/journals/phytomed

Phytomedicine

An in vitro evaluation of human cytochrome P450 3A4 inhibition by selected commercial herbal extracts and tinctures ]. W. Budzinski", B. C. Foster", S. Vandenhoek' and]. T. Amason! lOttawa-Carieton Institute of Biology, University of Ottawa, Ottawa, Ontario, Canada, 20ffice of Science, Therapeutic Products Programme, Health Canada, Ottawa, Ontario, Canada 3Digestive Diseases Research Group, University of Ottawa, Ottawa, Ontario, Canada

Summary Serial dilutions of 21 commercial ethanolic herbal extracts and tinctures, and 13 related pure plant compounds have been analyzed for their in vitro cytochrome P450 3A4 (CYP3A4) inhibitory capability via a fluorometric microtitre plate assay. Roughly 75% of the commercial products and 50% of the pure compounds showed significant inhibition of CYP3A4 metabolite formation. For each herbal product and pure compound exhibiting dose-dependency, the inhibition values were used to generate median inhibitory concentration (ICso) curves using linear regression. Among the commercial extracts, Hydrastis canadensis (goldenseal), Hypericum perforatum (St. John's wort), and Uncaria tomentosa (eat's claw) had the lowest ICso values at < 1% full strength, followed by Echinacea angustifolia roots, Trifolium pratense (wild cherry), Matricaria chamomilla (chamomile), and Glycyrrhiza glabra (licorice), which had ICso values ranging from 1%-2 % of full strength. Dillapiol, hypericin, and naringenin had the lowest IC so values among the pure plant compounds at < 0.5 mM; dillapiol was the most potent inhibitor at 23.3 times the concentration of the positive CYP3A4 inhibitor ketoconazole. Utilizing high-throughput screening methodologies for assessing CYP3A4 inhibition by natural products has important implications for predicting the likelihood of potential herbal-drug interactions, as well as determining candidates for further in-depth analyses. Key words: cytochrome P450, CYP3A4, inhibition, commercial herbal extracts and tinctures, potential risk, herbal-drug interactions.

Introduction The evaluation of herbal remedies with respect to drug disposition is an area that is not yet well-documented (De Smet and Brouwers, 1997). The area is of recent interest, since there has been a dramatic increase in the number of North Americans using herbal products (Brevoort, 1998), often combined with conventional drugs. Pharmacokinetic and other in vitro studies can provide information on indirect health risks and/or benefits potentially associated with the use of herbal remedies, which may include either a reduction or potentiation in the efficacy of concurrently used conventional medicines (De Smet, 1995). However, the trend

in increasing consumer demand for herbal products far exceeds the rate at which these products are being scientifically evaluated (Kozyrskyj, 1997), and the large number of available products precludes studies on all of them. Recently, several reports have demonstrated that concomitant oral administration of natural products may affect drug metabolism in humans (Bartle and Ferland, 1998; Foster et al., 1999; Nebel et al., 1999; Taylor and Wilt, 1999). The most widely studied natural product is grapefruit juice, which has been found to increase the bioavailabilty and/or prolong the metabolic elimina0944-7113/00107/04-273 $ 12.0010

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tion of many drugs, such as the dihydropyridine-type calcium channel blockers, the histamine-I receptor antagonist terfenadine, quinidine, the benzodiazepine rnidazolam, 17-~-estradiol, and caffeine (Ameer and Weintraub, 1997; Bailey et al., 1998), thereby significantly increasing their plasma concentrations. The specific mode of action of grapefruit juice appears to be the disruption of first-pass degradation detoxification reactions mediated primarily by the key xenobioticbiotransforming phase I enzyme cytochrome P450 3A4 (CYP3A4) (Ameer and Weintraub, 1997; Chan et al., 1998; Fuhr, 1998; He et al., 1998; Lown et al., 1997). Located in high amounts in the small intestinal epithelium and liver, CYP3A4 is a major contributor to the presystemic metabolism of drugs administered orally. Because CYP3A4 has an extremely wide substrate specificity and many in vitro and in vivo interactions involving bioactive compounds, it appears to mediate the oxidation of approximately 40-50% of the drugs administered to humans (Thummel and Wilkinson, 1998). Consequently, inhibition of CYP3A4 has important implications for possible drug-drug interactions, and it is logical to assume that natural products other than grapefruit juice may exhibit similar biological activities. As an early step towards assessing possible human exposure to adverse herbal product-drug interactions, the evaluation of the inhibitory potential of commercial herbal products against CYP3A4 is considered here. The rapid in vitro fluorometric microtiter plate assay allows assessments of many products in a short time and identifies possible candidates for herbal product-drug interactions that can be advanced to detailed in vivo studies.

officinalis); and wild cherry bark (Prunus serotina). Voucher samples have been retained at the University of Ottawa. A total of 21 different preparations were tested. Although all of the commercial herbal preparations contained ethanol (EtOH), the exact plant-material extraction procedures for each product was unknown. Therefore, the words "extracts" and "tinctures" are used synonymously to refer to anyone of the commercial herbal products. Subsamples (10 ml) from each herbal preparation were standardized to a total EtOH concentration of 55% and used as stock solutions. This concentration was chosen because the majority of the commercial products already contained this level of EtOH. Extracts with an EtOH concentration lower than 55% (U. tomentosa and T pratense -40%; A. lappa -31 %; and E. senticosus -10%) were adjusted to 55% EtOH using 95% EtOH (Commercial Alcohols Inc., Montreal, PQ). The Ginkgo leaf extract contained 66% EtOH and was diluted with distilled water to 55%. The EtOH concentration for P. quinquefolius and P. serotina was not indicated, and these two extracts were rotoevaporated to dryness with a PIAB-Labvac rotovapor, and subsequently rehydrated with 10 ml of 55% EtOH (prepared from 95% EtOH and distilled water). Prior to testing, standardized extracts were microfiltered through a Millex-Lf.R, 0.5 prn filter unit (Millipore, Bedford, MA). Serial dilutions were prepared by adding 100 ul of filtered and standardized-full strength extract to 100 pl of 55% EtOH in a 0.6-ml microfuge tube, and agitating the mixture for 10 seconds using a Fisher Scientific Vortex-Genie set to level 8. This procedure was repeated to yield 7 serial concentrations (100% -1.56%) relative to each extract's standardized full strength. All herbal stock solutions and dilutions were stored at room temperature in the dark.

Materials and Methods Commercial Plant Extracts and Tinctures

Pure Compounds

A variety of commercial-grade plant extracts and tinctures currently available in Canada were obtained, namely: burdock root (Arctium lappa); eat's claw (Uncaria tomentosa); chamomile (Matricaria chamomilla), devil's claw (Harpagophytum procumbens); Echinacea 1:1 blend (E. angustifolia and E. purpurea); Echinacea roots (E. angustifolia); Echinacea roots (E. purpurea); Echinacea tops (E. purpurea); elder fruit (Sambucus canadensis); feverfew (Tanacetum parthenium); Ginkgo leaf (G. biloba); goldenseal (Hydrastis canadensis); licorice (Glycyrrhiza glabra); milk thistle (Silybum marianum); North American ginseng (Panax quinquefolius); red clover herb and flower buds (Trifolium pratense); St. John's wort (Hypericum perforatum); saw palmetto (Serenoa repens); Siberian ginseng (Eleutherococcus senticosus); valerian root (Valeriana

A total of 13 pure herbal derived compounds (see Figure 1), all of which are well established phytochemical markers associated with several of the tested herbal extracts (Harborne and Baxter, 1993), were tested against CYP3A4 in vitro. Dillapiol was isolated formerly from Piper aduncum as described by Bernard et al. (1995), while chicoric acid and echinacoside were isolated previously from Echinacea spp. as outlined by Bergeron et al. (2000). Dihydrokaempferol, eriodictyol, and naringenin were isolated from Prunus serotina as described by Omar (2000). Isofraxidin-7-0-~-D-glucoside and Rg 1 ginsenoside were supplied by Harry Fong (University of Illinois). The remaining plant compounds were obtained from various chemical companies, as follows: harpagoside, parthenolide, and valerenic acid from Indofine Chemical Company Inc. (Belle Mead, NJ), hy-

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pericin from Vimrx Pharmaceuticals (Irvine, CAl, and berberine sulfate from SB Penick and Co. Botanical Products and Fine Chemicals (New York, NY). The antifungal drug ketoconazole was acquired from Janssen Pharmaceutica (Lammerdries, Belgium). All pure compounds were crystalline or powdered solids, with the exception of dillapiol, which was an oily liquid. Purity (> 95 %) was assured by HPLC. All other chemicals and solvents were of analytical grade. Solutions (:5 5 ml) were prepared by dissolving the pure compound in 55% EtOH and vortexing the mixture for 1 min, with a subsequent I-min sonication (Branson 2200 Sonicator, Shelton, CT). Starting concentrations were based on the results of a preliminary screen against CYP3A4 (n =1) involving 3 dilutions (1 mg/ml, 0.1 mg/ml, and O.Ol-mg/ml; results not shown). Dilutions were performed in the same manner as described in "Commercial Plant Extracts and Tinctures". Dillapiol, echinacoside, hypericin, naringenin, and parthenolide dilutions were prepared from 1 mg/ml to 15.6 ug/ml, Chicoric acid and eriodictyol serial dilutions were prepared from 2 mg/ml to 31.3 pg/ml, Berberine sulfate, dihydrokaempferol, harpagoside, isofraxidin-7-0-~-D-glucoside, Rg . ginsenoside, and valerenic acid serial dilutions were prepared from 4 mg/ml to 62.5 ug/rnl. Serial dilutions of the positive control CYP3A4 inhibitor ketoconazole were prepared from 4 ug/ml to 62.5 pg/ml.

Assay Procedures Pure plant compounds and commercial alcoholic plant extracts and tinctures were screened for their ability to inhibit CYP3A4 in vitro using a fluorometric microtitre plate assay. The procedure used was adapted and modified from that reported by Crespi et al. (1997) and later described by GENTEST Corporation (GENTEST, 1998). Briefly, assays were performed in clear-bottom, opaque-welled microtitre plates (96 well, Corning Costar, model # CSOO-3632, Corning, NY) . Wells were designated as " Control" , "Blank", "Test" or "Test-Blank". Control wells consisted of 55 % EtOH and NADPH (~-nicotinamide adenine dinucleotide phosphate, reduced form; Sigma Chemical Co., S1. Louis, MO) solution; blank wells consisted of 55 % EtOH and buffer solution; test wells consisted of the extract or pure compound at a particular concentration and NADPH solution; and test-blank wells consisted of the corresponding extract or pure compound concentration and buffer solution. Enzyme solution was added to all wells. Solutions were added to the appropriate designated wells in the following order and quantity: 95 III of buffer or NADPH solution,S pi of 55% EtOH or test solution, and 100 III of enzyme solution. Buffer solu-

tion (pH 7.4) consisted of an approximate 1:4.7 (v/v) mixture of 0.5 M potassium phosphate dissolved in distilled water. NADPH was mixed with buffer to yield a 15 mg/ml solution and stored at -4 °C for up to 2 weeks before use. Prior to addition, NADPH solution was thawed and further diluted with buffer to 1.07 mg/m!. Fifty-five percent EtOH was made by diluting the 95 % EtOH with distilled water. An enzyme stock solution (760 pl/rnl buffer, 200 pl/ml distilled water, 10 ul/rnl substrate solution, and 30 ul/rnl enzyme) was made by pre-warming the water/buffer mixture to 37°C for 10 min in a sand bath, adding the substrate solution of 1.5 mg/ml 7-benzyloxyresorufin (lot # 27H4119; Sigma Chemical Co., St. Louis, MO) in acetonitrile, vortexing the mixture for 5 seconds, and adding rapidly thawed CYP3A4 (300 pmoles, CYP3A4+0R+b s SUPERSOMES, GENTEST, lot # 11; Woburn, MA). Enzymes were stored at -80°C until used and were not subjected to more than 2 freezethaw cycles. Once all of the solutions were added sequentially (buffer or NADPH solution, EtOH or herbal test mixture, enzyme solution) to the designated wells, plates were incubated for 1 hat 37°C. After incubation, 100 pi of stop solution consisting of a 1:4 (v/v) acetonitrile to 0.5M Tris base (Tris-[hydroxymethyl]aminomethane; Sigma Chemical Co., St. Louis, MO) in distilled water (pH 9) was added to each well , and plates were agitated subsequently on a Lab-Line Instruments Inc. Titer Plate Shaker (Melrose Park, IL) for 10 s at setting 2. A Millipore Cytofluor 2350 Fluorescence Measurement System (Bedford, MA) set to a 530 nm (30 nm bandwidth) excitation filter and a 590 nm (35 nm bandwidth) emission filter was used to analyze each plate. Readings were obtained for a single pass with sensitivity settings set to 4, 3, and 1, respectively. Only data sets yielding the highest readings without saturation were used to calculate percent inhibition values for each level of the extract or pure compound dilutions. Percent inhibition calculations were based on differences in fluorescence between the control/blank wells and test/test blank wells. For each assay, no more than one dilution set for either the plant extracts or pure compounds was performed simultaneously on the same plate during a single experiment. Therefore, a control value was assessed ever y time an assay was performed and was based upon the mean difference between each control and blank well (n = 3). Three experimental values were achieved for each level of the dilution in the same manner. Test and test-blank wells were performed in triplicate for all dilutions of the plant extracts. Only one set of testblanks at the highest concentration (n = 3), however, was used for colorless pure compound solutions (dihydrokaempferol, dillapiol, echinacoside, harpagoside,

An in vitro evaluation of human cytochrome P450 3A4 inhibition isofraxidin-7 -O-~- D-glucoside, ketoconazole, naringenin, parthenolide, Rg 1 ginsenoside, and valerenic acid). All assays were performed under gold fluorescent lighting (Industrial Lighting, Ottawa, ON).

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Statistical Analysis Percent inhibition values for each assay were plotted against the log-transformed concentration (% full strength for plant extracts and tinctures, and mg/ml or pg/rnl for pure compounds) and analyzed using simple linear regression with SYSTAT software (v. 7.0.1. for Windows, SPSS Inc., Chicago, IL). Only percent inhibition values> 2% and < 98% were considered valid for regression analysis, since points lying beyond this range were assumed to represent no-effect (0% inhibition) or complete enzyme saturation (100% inhibition), respectively. Mathematically calculated inhibition values < 0% and> 100% were observed for some of the tested products at varying dilution concentrations, and these too were considered as no-effect or saturation values, respectively. Median inhibitory concentration (IC so) values were obtained for dilutions that exhibited clear dose-dependency (a one-tailed p-value < 0.05) by the formula: y = mx + b; where (y) is the percent inhibition, (x) is the relative concentration, (m) is the slope, and (b) is the constant, given by the linear regression analysis.

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Commercial Plant Extracts and Tinctures Serial dilutions of standardized commercial extracts and tinctures were analyzed for their ability to inhibit CYP3A4-mediated metabolism of the test substrate 7benzyloxyresorufin. Figure 2a depicts a representative IC so regression line for a herbal extract (1:1 E. angustifolia/purpurea blend), while the relevant statistical values for the regression curves of all of the commercial products are summarized in Table 1. Regression analysis assumptions were tested and met for all of the evaluated products, with the following exceptions: E. purpurea root, licorice root, and devil's claw extracts were found to be non-normally distributed due the presence of a single outlying point (verified by eliminating the outlier and re-running the analysis; data not shown). For these products, normality was assumed. Of the 21 selected products, only 3 extracts were found to be non-inhibitory within the tested range (p > 0.05): E. senticosus, P. quinquefalius, and H. pracumbens. The remaining products were ranked according to their calculated IC so values. Products with the lowest IC so values were ranked the most inhibitory. An IC so value could not be determined for A. lappa or T. parthenium,

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because full-strength trials of these extracts failed to inhibit at least 50% of the CYP3A4 in this assay. These two extracts clearly did have a very low inhibitory action, since dose-dependency was observed (p < 0.05). Seven of the preparations were found to be moderately inhibitory (IC so values > 5% and < 10% full strength): 1:1 E. angustifolia/purpurea blend, E. purpurea tops, P. seratina, S. canadensis, S. repens, S. marianum, and V. officinalis. Of this group, V. affici-

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Table 1. The median inhibitory concentration (ICso) values for commercial plant extracts and tinctures against cytochrome P4503M. Regression Line:

rc., Relative Commercial Extractffincture

Arctium lappa

Concentration (% Full Strength)

Slope

> 100

18.88 (14.66,23.10) 35.15 (32.40,37.90) 24.85 (20.17,29.52) 34.81 (30.34,39.29) 43.75 (40.40,47.10) 7.78 (-2.25, 17.81) 69.38 (53.09,85.68) 43.95 (32.68,55.22) 0.14 (-3.71,3.99) 15.02 (11.05,18.99) 17.33 (12.38,22.27) 21.64 (19.90,23.32) -3.96 (-12.12,4.20) 77.47 (7020,84.74) 26.24 (20.73,31.75) 38.93 (34.17,43.68) 38.45 (35.20,41.69) 22.14 (15.82,28.46) 29.38 (24.00,34.76) 80.28 (31.81,128.75) 19.08 (13.79,24.37)

Echinacea angustifolialpurpurea 6.73 a (1:1)

Echinacea angustifolia roots Echinacea purpurea roots Echinacea purpurea tops Eleutherococcus senticosus Ginkgo biloba Glycyrrhiza glabra Harpagophytum procumbens Hydrastis canadensis Hypericum perforatum Matricaria chamomilla Panax quinquefolius Prunus seratina Sambucus canadensis Serenoa repens Silybum marianum Tanacetum parthenium Trifolium pratense Uncaria tomentosa Valeriana officinalis

(10.09,4.75) 1.05 b (2.19,0.64) 3.99 a (7.74,2.39) 8.56 a (13.05,5.95) NI 4.75 a (12.82,2.57) 1.83 (4.29,1.11) NI 0.03 b (0.02,0.04) 0.04 b (0.03, 0.05) 1.48 a (1.97,1.16) NI 6.90 a (10.45,4.89) 6.82 a (24.41,2.97) 7.41" (14.39,4.41) 5.22 a (7,94, 3.67) > 100 1.05 b (1.80,0.72) 0.79 b (1.56,0.66) 9.56 a (70,49, 3.09)

Constant

N

R2

P

Ranked Inhibition

(1 tail) 21 9.53 (425,14.81) 21 20.91 (17.47,24.34) 18 49.43 (43.12,55.73) 18 29.07 {23.04, 35.11) 20 9.218 (4.93,13.51) 5.74 17 (-7.77,19.24) 12 3.04 (-8.82,14.90) 12 38.45 (29.33,47.57) 21 25.23 (20.41,30.05) 16 72.80 (68.80, 76.80) 14 74.01 (69.78, 78.25) 21 46.32 (44.13,48.51) 17 20.53 (9.54,31.52) 15 -14.97 (-21.53, -8.41) 21 28.12 (21.23,35.01) 20 16.15 (10.43,21.87) 21 22.39 (18.33,26.45) 18 -6.19 (-14.57,2.18) 17 49.42 (43.89,54.96) 4 58.37 (43.88,72.86) 20 31.30 (24.52,38.08)

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16

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7

0.977

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14

0.154

0.060

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0.900

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8

0.883

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6

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0.470

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An in vitro evaluation of human cytochrome P450 3A4 inhibition

nalis was the weakest inhibitor and had a high variability with a potential lC 50 value range of 3.09%-70.49%, given by the 95% confidence interval. Similarly, six preparations had a relatively high inhibitory ability (ICso values > 1% and < 5% full strength): E. angustifolia roots, E. purpurea roots, G. biloba, G. glabra, M. chamomilla, and T. pratense. Finally, the most potent inhibitors of CYP3A4 in this assay (ICso values < 1% full strength) were: H. canadensis, H. perforatum, and U. tomentosa. Based on the determined lC so values, H. canadensis extract was determined to be the most potent enzyme inhibitor. It is important to note that the preparation of U. tomentosa selected for this trial, may however, in fact be a more potent inhibitor since the determined lC so value for this product was based only on a few readings (N 4) and may be highly conservative. Within the selected experimental protocols for the dilution of the commercial tinctures, U. tomentosa was found to give inhibition readings> 100 % (saturation of the enzyme) for relative concentrations ~ 3.13%.

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Pure Compounds

Serial dilutions of various pure compound solutions were also analyzed for their ability to inhibit CYP3A4. Figure 2b depicts a representative lC so regression line for a pure compound (naringenin), while the relevant statistical values for the regression curves of all of the pure compounds are summarized in Table 2. As expected, the CYP3A4 inhibitor ketoconazole had the lowest IC so value (7.18X10-4 mM) and was found to be 23.3 times more inhibitory than the most inhibitory plant phytochemical (dillapiol: 1.67X 10-2 mM). Of the 13 plant compounds, 2 were found to be non-inhibitory within the tested range (p > 0.05): harpagoside (from H. procumbens), and isofraxidin-F-Ocf-Deglucoside (from E. senticosus). Isofraxidin-7-0-(3-D-glucoside failed to yield any inhibition values > 1% for any of the dilutions, and thus no regression analysis was performed. The remaining compounds were inhibitory to varying degrees and were also ranked according to their calculated lC so values. Compounds with the lowest lC so values were ranked the most inhibitory. Compounds with undetermined IC so values were ranked together (see Table 2). Four of the compounds were considered to have a very low inhibitory ability against CYP3A4 in this assay: chicoric acid (from E. angustifolia), eriodictyol (from P. serotina), Rg 1 ginsenoside (from P. quinquefolius), and valerenic acid (from V. officinalis). ICso values for these compounds were not achieved within the experimental regime and were not extrapolated beyond the maxi mum tested concentration, since the observed dose-dependency was also low (m < 20); predicted lC so values

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would therefore have been highly inaccurate with a large amount of variability. Three of the pure compounds were found to have a relatively moderate inhibitory ability (ICso values> 5 mM and < 10 mM): berberine (from H. canadensis), echinacoside (from Echinacea spp.), and dihydrokaempferol (from P. serotina). Parthenolide (from T. parthenium) was the only compound exhibiting relatively high inhibition (ICso values > 1 mM and < 5 mM). Three of the compounds were found to be very high relative inhibitors of CYP3A4 (IC50 values < 1 mM): dillapiol (from Piperaceae and Anethum sp.), hypericin (from H. perforatumi, and naringenin (from Citrus sp. and P. serotina). Of the three compounds, dillapiol was the most potent inhibitor of CYP3A4 with an IC so value 23.3 times greater than that of ketoconazole.

Discussion We have demonstrated clearly that approximately 66% of the selected herbal preparations were significantly inhibitory of CYP3A4-mediated metabolism of 7-benzyloxyresorufin, with lC so values occurring at relative concentrations less than 10 % of the full strength preparations. Natural products which yield higher levels of CYP3A4 (or other isozyme) inhibition from typical high-throughput screens performed in this manner are arguably better candidates for further in vivo work and subsequent clinical testing. IC so value determinations can be used to gain preliminary insight and aid in establishing and classifying at -risk herbals potentially capable of displaying adverse drug-drug interactions when taken concomitantly with prescription or conventional non-prescription medicines metabolized by the same isozyme. For example, based on the assay results, one could surmise that ethanolic extracts of H. perforatum and H. canadensis may potentially pose a substantial health risk to individuals when taken along with conventional medicines that are also metabolized by CYP3A4, because these extracts achieved very low lC so relative concentrations « 1% ) when present at only 2.5 % of the total reaction volume. Equally, one could surmise that ethanolic extracts of T. parthenium and A. lappa are less likely to pose as great a health risk under the same conditions, although the possibility still exists since dose-dependency was observed. With respect to the lC so values obtained for some of the related pure compounds, it is important to note that no direct quantitative comparisons were made with the commercial herbal extracts, even if the levels of these marker compounds had been determined. Because of the large number of substances in these preparations, it is difficult to identify and quantify all poten-

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Table 2. The median inhibitory concentration (ICso) values for pure compounds dissolved in 55 % ethanol against cytochrome P4503M. Regression Line: Compound ICso Concentrat ion (mM) Berberine Chicoric Acid (Cichoric Acid) Dihydrokaempferol (Aromadendrin) Dillapiol Echinacoside Eriodictyol Harpago side Hypericin Isofraxidin-7-0 -~-D-

Slope

Constant

5.72' (7.59,4.53) > 4.22

19.85 (17.97, 21.72) 10.39 (7.56, 13.22) 5.79" 43.7 8 (7.23, 4.86) (39.06, 48.50) 1.67xI0-2b 20.85 (1.07X 10-2,2.35x I 0-2) (18.26, 23.45) 6.29 b 13.77 (71.56, 2.07) (8.65,18 .90) » 6.94 3.51 (-0.37, 7.39) NI - 7.10 (-9.93, -4.28) 0.33" 35.08 (0.46, 0.28 ) (24.38, 45.77) NI *

glucoside (Eleutheroside Bl ) Naringenin 0.42 a (0.43, 0.40 ) 1.36a Partheno lide (1.87, 1.09) Rg1 Ginsenoside » 8.09

N

40.03 21 (38.77,41.29) 31.59 20 (29.31,33.86) 40.25 18 (37.57, 42.93) 100.70 21 (97.88, 103.52) 21 40.43 (34.86,45.99) 17.16 19 (13.87, 20.44) 10.83 21 (8.93, 12.74) 77.30 18 (65.45,89.15 ) * "

28.24 21 76.74 (25.11, 31.36) (73.35, 80.13) 23.66 21 61.14 (19.31, 28.01) (56.41, 65.86) 9.82 17.98 16 (2.42, 17.22) (13.59, 22.36) 14.30 21 19.54 (6.84, 21.76) (14.52, 24.56)

Valerenic Acid

> 17.07

Ketoconazole

7.18 X10-4a 34.34 (7.47xI0-4,6.97X I 0-4) (29.97,38.70)

64.36 18 (62.02,66.70)

Ranked Inhibition

R2

P (1 tail)

0.963

0.000

6

0.768

0.000

9

0.960

0.000

7

0.937

0.000

2

0.625

0.000

8

0.177

0.037

9

0.593

1.000

NI

0.751

0.000

3

"

*

0.950

0.000

4

0.872

0.000

5

0.366

0.007

9

0.459

0.001

9

0.946

0.000

1

NI

Note: Numbers in brackets correspond to the lower and upper 95% confidence limits of the particular value respectively. * analysis not performed. a value was achieved within the tested range. b value was achieved by extrapol ating the regression line beyond the tested range. NI - non inhibitory within the tested range.

tial compounds and in teractions in a given extract. For example, dillapiol, found to be the most potent inhibitor of CYP3A4 among the pure plant co mpounds screened in this study, with an IC so value (1.67xlO-2 mM) 23 .3 times higher than that of the positive control inhibitor ketoconazole, has been demonstrated to be an effective synergist of several insecticides, including carbaryl and pyrethrin. This synergistic role relates directly to its enzyme-inhibiting action against insect cytochrome P450-de pendent polysubstrate monooxygenases (PSMOs) (Bernard et al., 1990; Bernard and Philogene, 1993). It is plausible that simi lar additive/ syne rgistic or antagonistic effects could occ ur in crude commercial herbal prep arations, thereby ex acerbating

the in hibitory effect of other compounds against CYP3A4. This further illustrates the importance of un dertaking a more thorough analysis of herbal products shown preliminarily to strongly interact with in vitro drug-metabolizing systems such as CYP3A4 microsomes. Undoubtedly, all in vitro drug interactions may be of clinical importance and it therefore becomes paramount to understand how the redirection and change of metabolic profiles by herbal products ca n affect the safety and efficacy of various drugs or oth er herbal products. In general, CYP3A4-mediated me ta bo lism of an affec ted drug must represent a major pathway for the product, and the magnitude of inhi bition must pro -

An in vitro evaluation of human cytochrome P450 3A4 inhibition duce a significant alteration in the drug's plasma concentration-time profile relative to the concentration-response relationship. Clinically important drug interactions generally occur only with drugs having a narrow and/or steep concentration-response relationship when co-administered with a potent inhibitor (Thummel and Wilkinson, 1998). Hence, despite the inability to determine pharmacokinetic values for crude herbal extracts, potentially clinically relevant CYP3A4 inhibition reactions can still be assessed from this type of screen on the basis of IC so value determination: the lower the determined IC so value of a given product, the higher its risk potential for generating adverse drug interactions when taken concomitantly with conventional medicines metabolized by the same isozyme. The distinction in classifying and assessing herbal extracts in this manner is consequently one of probability in identifying the likelihood of an interaction between a herbal product and a concomitantly administered conventional drug or other herbal product. The potential for generating adverse drug reactions increases with repeated product use alone, or in combination with other natural health products or therapeutic agents. The effect of repeated use of herbal products on drug disposition is not known, but caution is advised as it is not unlikely that these products could also become enzyme inducers. To the best of our knowledge, at the time of these studies, there have been no other reports in the literature examining the inhibitory potential of commercial herbal extracts or tinctures against CYP3A4. Investigating herbal product inhibition of this enzyme is of great importance because such bioactivity could be an indicator of conceivable potentiation effects of herbal phyrochernicals with conventional drug therapies. Implications of herbal CYP3A4 inhibition are twofold: (1) herbal products shown to inhibit CYP3A4 could be used as drug-sparing agents when taken concomitantly with conventional medicines, thereby decreasing the dosage and financial costs of expensive drug regimes; (2) herbal products shown to inhibit CYP3A4 could potentially increase the toxicity levels of concomitantly administered conventional drugs due to prolonged drug residence time within the body resulting from decreased metabolic breakdown. The former can only be achieved if the pharmacokinetics of standardized herbal products, in combination with extensive therapeutic drug monitoring, can be realized and maintained. Further CYP3A4 and other cytochrome P450 isozyme in vitro work is required to determine if the activity of the commercial herbal products can be predicted by the titer of marker compounds they contain. Finally, clinical studies involving detailed pharmacokinetics for numerous key conventional drugs in the presence or absence of these herbal extracts, especially

281

those found to be potent CYP3A4 inhibitors, must be completed to determine if the present in vitro results are relevant in vivo. Acknowledgements

We would like to thank Dr. Harpal Buttar and Ms. Amanda Mills for their thorough review of this manuscript. This research was supported by the AIDS program committee of Ontario and the National Science and Engineering Research Council (Strategic Program).

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Address

J. T.

Amason, Ottawa-Carleton Institute of Biology, Department of Biology, University of Ottawa, 30 Marie Curie St., PO Box 450, Stn. A, Ottawa, ON, KlN 6N5, Canada. Phone: (613)-562-5262; Fax: (613)-562-5765; E-mail: [email protected]